Dimethyl (p-methoxybenzylidene)malonate

Endpoint summary

Description of key information

Skin irritation: Only slight, reversible irritating effects were observed in a reliable study, which did not require classification.

Eye irritation: A reliable study observed eye irritating effects in rabbits, which require classification. No predíction of eye irritation was possible from a reliable Bovine Corneal Opacity and Permeability (BCOP) assay.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Remarks:

SPF

Details on test animals or test system and environmental conditions:

Age at start of treatment: 14-15 weeks (male), 13-14 weeks (females)
Body weights at start of treatment: 2.3 - 2.9 kg
Identification: By unique cage number and corresponding ear number.
Acclimatization: Six days under laboratory conditions after health examination. Only animals without any visual signs of illness were used fior the study.
Allocation: Male No. 88, Female Nos. 89 and 90

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

water

Controls:

not required

Amount / concentration applied:

The test item was administered at 0.5 g/animal, the dose specified in the test guidelines for a solid test item.

Duration of treatment / exposure:

4 hours

Observation period:

The skin irritation was assessed according to the numerical scoring system listed in the EEC Commission Directive 92/69/EEC, July 31, 1992 at approximately 1, 24, 48 and 72 hours, after the removal of the dressing, gauze patch and test item.

Number of animals:

1 male and 2 females

Details on study design:

TEST ITEM PREPARATION:
0.5 g (per animal) of the test item was weighed as delivered by the sponsor and then moistened with bi-distilled water before application. According to Directive 92/69 EEC, B.4 and OECD Guidelines 404, a substance needs not to be tested if the pH-value is less than 2 or greater than 11.5, owing to its predictable corrosive properties. Prior to the application the pH of the test item was determined at a concentration of 1 % in water with pH-Test-Strip (E. Merck, D-64271 Darmstadt / Germany). The pH was found to be 6.

TREATMENT:
Four days before treatment, the left flank was clipped with an electric clipper, exposing an area of approximately 100 cm² (10 cm x 10 cm). The skin of the animals was examined one day before treatment, and regrown fur of all animals was again clipped. Animals with overt signs of skin injury or marked irritation which may have interfered with the interpretation of the results were not used in the test. On the day of treatment, 0.5 g of test item was placed on a surgical gauze patch (ca. 2.5 cm x 2.5 cm). This gauze patch was applied to approximately 6 cm² of the intact skin of the clipped area. The patch was covered with a semi-occlusive dressing [Isoelast Heftpflaster Acryl 8 x 75 cm]. The dressing was wrapped around the abdomen and anchored with tape. The duration of treatment was 4 hours. Then the dressing was removed and the skin was flushed with lukewarm tap water to clean the application site so that any reactions (erythema) were clearly visible at that time.

OBSERVATIONS:

Viability/Mortality: Daily from delivery of the animals to the termination of test.
Clinical signs: Daily from delivery of the animals to the termination of test.
Body weights: At start of acclimatization, on the day of application and at termination of observation.

Irritation parameter:

other: cumulative (erythema and oedema score)

Basis:

mean

Time point:

other: 1 h

Score:

1.33

Max. score:

2

Reversibility:

not specified

Irritation parameter:

other: cumulative (erythema and oedema score)

Basis:

mean

Time point:

24 h

Score:

0.67

Max. score:

1

Reversibility:

not specified

Irritation parameter:

other: cumulative (erythema and oedema score)

Basis:

mean

Time point:

48 h

Score:

0

Max. score:

0

Reversibility:

not specified

Irritation parameter:

other: cumulative (erythema and oedema score)

Basis:

mean

Time point:

72 h

Score:

0

Max. score:

0

Reversibility:

not specified

Irritation parameter:

erythema score

Basis:

mean

Remarks:

animal #1, #2, #3

Time point:

24/48/72 h

Score:

0.22

Max. score:

4

Reversibility:

fully reversible within: 48 h

Remarks on result:

no indication of irritation

Irritation parameter:

edema score

Basis:

mean

Remarks:

animal #1, #2, #3

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable since no effect

Remarks on result:

no indication of irritation

VIABILITY/MORTALITY/CLINICAL SIGNS:

No clinical signs of systemic toxicity were observed in the animals during the study and no mortality occurred.

IRRITATION:

Application of the test item to healthy intact rabbit skin resulted in a primary irritation score of 0.22.

Local signs (mean values from 24 to 72 hours) consisted of grade 0.22 erythema and grade 0.00 oedema.

A very slight erythema was observed in all animals at the 1 -hour observation before disappearing by 24 hours (one animal) or 48 hours (two animals). A very slight oedema was noted in one animal at the 1 -hour reading.

COLORATION:

No staining by the test item of the treated skin was observed.

CORROSION:

No irreversible alterations of the treated skin were observed nor were corrosive effects evident on the skin.

BODY WEIGHTS:

The body weights of all rabbits were considered to be within the normal range of variability.

Interpretation of results:

other: not irritating

Conclusions:

Based upon the referred classification criteria (EEC Commission Directive 93/21/EEC of April 27, 1993 and Regulation (EC) No. 1272/2008 (CLP)), the test item is considered to be "not irritating" to rabbit skin.

Executive summary:

The primary skin irritation potential of the test item was investigated by topical semi-occlusive application of 0.5 g to 6 cm² intact left flank of each of three young adult New Zealand White rabbits. The duration of treatment was four hours. The scoring of skin reactions was performed 1, 24, 48 and 72 hours after removal of the dressing. The scores of each animal at the following reading times (24, 48, 72 hours) were used in calculating the respective mean values for each type of lesion.

The primary irritation score was calculated by totaling the individual cumulative scores at 24, 48 and 72 hours and then dividing by the number of data points. The primary irritation score was 0.22 (max. 8.0).

Local signs (mean values from 24 to 72 hours) consisted of grade 0.22 erythema and grade 0.00 oedema.

A very slight erythema was observed in all animals at the 1 -hour observation before disappearing by 24 hours (one animal) or 48 hours (two animals). A very slight oedema was noted in one animal at the 1 -hour reading.

The test item caused no staining of the treated skin.

No corrosive effects were noted on the treated skin of any animal at any measuring interval.

Based upon the referred classification criteria (EEC Commission Directive 93/21/EEC of April 27, 1993 and Regulation (EC) No. 1272/2008 (CLP)), the test item is considered to be "not irritating" to rabbit skin

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-06-06 to 2017-09-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)

Specific details on test material used for the study:

Name: Hostavin PR-25 P
Chemical Name: Propanedioic acid, [(4-methoxy-phenyl)-methylene]-,dimethyl ester
Product (Common Name/Code): Hostavin PR-25 P
CAS No.: 7443-25-6
Batch No.: CHA0122531
Molecular Weight: 250 g/mol
Physical State: solid
Colour: white
Density: 1.348
Purity: 99.8%
Date of Analysis: 27 January 2017
Expiry Date: 15 March 2018
Storage Conditions: room temperature, protected from light
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

Test System
Species/strain: healthy New Zealand White Rabbits, Crl: KBL (NZW)
Source: Charles River Deutschland, 97633 Sulzfeld, Germany
Sex: female
Body weight at the
beginning of the study: animal no. 1: 4.8 kg
animal no. 2: 4.6 kg
Age at the
beginning of the study: animal no. 1: approximately 29 weeks old
animal no. 2: approximately 31 weeks old
Number of animals: 2
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to Art. 9.2, No. 7 of the German Act on Animal Welfare the animals were bred for experimental purposes. 
This study was performed in an AAALAC-accredited laboratory. According to German animal protection law, the study type has been reviewed and accepted by local authorities. Furthermore, the study has been subjected to Ethical Review Process and was authorised by the Bavarian animal welfare administration.

Housing and Feeding Conditions
- Semi barrier in an air-conditioned room
- Temperature: 18 +/- 3 °C (recommendations of TVT, GV-SOLAS)
- Relative humidity: 55 +/- 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: at least 10 x / hour
- Free access autoclaved hay and to Altromin 2123 maintenance diet for rabbits, rich in crude fibre
- Free access to tap water (drinking water, municipal residue control, microbiological controls at regular intervals)
- Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich
- Housed in ABS-plastic or Noryl rabbit cages, floor 4200 cm2
- Adequate acclimatisation period (at least 5 days) under laboratory conditions

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

A dose of 0.1 g of the test item was applied to the test site.

Duration of treatment / exposure:

The test item was applied at a single dose in the conjunctival sac of one eye of each test animal after pulling the lower lid away from the eyeball.
The lids were then gently held together for about 1 second in order to prevent loss of the material. The untreated contralateral eye served as control.
The treated eye of animal no. 1 was not rinsed after the application. The treated eye of animal no. 2 was rinsed with physiological saline 0.9% NaCl 1 hour after the application due to residues of the test item at the animals eye.

Observation period (in vivo):

The animals were observed for 72 hours after dosing. To determine the reversibility of the observed effects, the observation period was extended up to 8 days after dosing.

Number of animals or in vitro replicates:

The in vivo test was performed initially using one animal.
The results of the initial test did not indicate the test item to be corrosive but a severe irritant to the eye using the procedure described.
In order to confirm the response, one additional animal was treated in the same manner.
According to OECD 405, section 23, treatment of a third animal was omitted as results from the second animal were sufficient to allow for hazard classification determination.

Details on study design:

Preparation of the Animals
Within 24 hours before the test and immediately prior to the application both eyes of each animal were examined. A health inspection was performed to ensure the good state of health of the animals.
Approximately 16 hours (animal no. 1) and 15 hours (animal no. 2) before the application the eyes were examined with the aid of a fluorescein solution (Fluoreszein SE Thilo, Alcon Pharma, lot no. H 603, expiry date: 08/2018). The eyes were rinsed with physiological saline 0.9% NaCl (Alleman Pharma, lot no. 609709, expiry date: 08/2019) after the examination.
None of the animals showed eye irritation, ocular defects, or pre-existing corneal injury.

Application
One hour before the application of the test item, 0.01 mg/kg of buprenorphine (Reckitt Benckiser, lot no.: 5481, expiry date: 01/2018 (animal no. 1) and lot no.: 5510, expiry date: 09/2018 (animal no. 2)) was administered subcutaneously in order to achieve a therapeutic level of systemic analgesia.
Approximately 5 minutes prior to the application of the test item, 1-2 drops of an ocular anaesthetic (proparacaine hydrochloride ophthalmic 0.5% solution, URSAPHARM, lot no.: 286171, expiry date: 07/2017) were administered in both the treated and the control eye of each animal.
The test item was applied at a single dose in the conjunctival sac of one eye of each test animal after pulling the lower lid away from the eyeball. The lids were then gently held together for about 1 second in order to prevent loss of the material. The untreated contralateral eye served as control.
The treated eye of animal no. 1 was not rinsed after the application. The treated eye of animal no. 2 was rinsed with physiological saline 0.9% NaCl 1 hour after the application due to residues of the test item at the animals eye.
To prevent pain and distress after the application of the test item both animals were treated with the following doses of buprenorphine and meloxicam:

Clinical Observation
The eyes were examined for signs of irritation throughout the observation period. The eye irritation was scored and recorded according to the Grading System for Ocular Lesion.
For the calculation only the 24, 48 and 72-hour readings were used.
72 hours post-application as well as at the end of the prolonged observation period the treated eyes were examined with the aid of a fluorescein solution. The eyes were rinsed with physiological saline 0.9% NaCl after the examination.

Body Weight
The animals were weighed prior to the administration and at the end of the observation period.

Evaluation of Results
The animal’s reactions were recorded according to the scoring system described at each time of observation.
For the calculation only the 24, 48 and 72-hour readings were used.
Nature, severity and duration of clinical observations were described.
The body weight changes were summarised in a tabular form.

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0.67

Max. score:

2

Reversibility:

fully reversible within: 8 days

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable since no effect

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2.33

Max. score:

4

Reversibility:

fully reversible within: 8 days

Remarks on result:

positive indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.67

Max. score:

3

Reversibility:

fully reversible within: 8 days

Remarks on result:

no indication of irritation

Irritation parameter:

iris score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

2

Reversibility:

other: not applicable since no effect

Remarks on result:

no indication of irritation

Irritation parameter:

cornea opacity score

Basis:

animal #2

Time point:

24/48/72 h

Score:

0

Max. score:

4

Reversibility:

other: not applicable since no effect

Remarks on result:

no indication of irritation

Irritation parameter:

conjunctivae score

Basis:

animal #2

Time point:

24/48/72 h

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 7 days

Remarks on result:

no indication of irritation

Irritation parameter:

chemosis score

Basis:

animal #2

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 7 days

Remarks on result:

positive indication of irritation

Irritant / corrosive response data:

After the application into the eyes of two female NZW rabbits the test item produced irritant but not corrosive ocular effects.
Neither mortality nor significant clinical signs of toxicity but local effects were observed.
At the end of the prolonged observation period no corneal lesions were found.
Conjunctival redness, chemosis and hypersecretion were observed and are described in the report.
Clinical findings and local changes (hypersecretion with moistening of the lids and surrounding hair, hypersecretion with wetting of the eyelids and hair around the eyes and clear flow secretion) were observed.

Other effects:

The body weight development of animal no. 1 was within the expected range. For animal no. 2 a moderate weight loss due to reduced food and water intake was observed

Interpretation of results:

Category 2 (irritating to eyes) based on GHS criteria

Conclusions:

Under the conditions of the present study, a single ocular application of the test item Hostavin PR-25 P to rabbits at a dose of 0.1 g produced irritant effects (Chemosis > 2), which were fully reversible within 8 days.
Neither mortalities nor significant clinical signs of toxicity but local effects were observed.

Executive summary:

Summary Results

Species/strain: New Zealand White Rabbits Crl: KBL (NZW)

Number of animals: 2

Amount of substance: 0.1 g per test site

First time of effects: animal no. 1: 1 hour post-application redness grade 2, chemosis grade 3, 24 hours post-apllication hypersecretion grade 1

animal no. 2: 1 hour post-application redness grade 1, chemosis grade 2

Last time of effects: animal no. 1: 48 hours post-application hypersectretion grade 1, 72 hours post-application chemosis grade 1 and 7 days post-application redness grade 1

animal no. 2: 5 days post-application chemosis grade 1 and 6 days post-application redness grade 1

Reversibility of the observed effects:  the changes were fully reversible within 8 days post-application

Method: OECD 405, EC 440/2008, Method B.5, OPPTS 870.2400

Conclusion

Under the conditions of the present study, a single ocular application of the test item Hostavin PR-25 P to rabbits at a dose of 0.1 g produced irritant effects, which were fully reversible within 8 days. Neither mortalities nor significant clinical signs of toxicity but local effects were observed.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In a reliable, GLP-compliant study according to OECD guideline, there were eye irritating effects

(mean scores of conjunctival redness: 1.3, cornea opacity: 0.0, iris effects: 0.3, chemosis score 2.2, which were fully reversible within 8 days)

requiring classification according to Regulation (EC) No. 1272/2008 (CLP).

Justification for classification or non-classification

Based on the observed eye irritation in an in vivo study classification according to Regulation (EC) No 1272/2008 as Eye Irrit. 2 is required. No classification for skin irritation/corrosion is necessary.